A major effort of our laboratory this past year was directed at characterizing the mechanisms underlying the trafficking of glutamate receptors and related proteins to the postsynaptic density. Much of the research has focused on NMDA receptors and their associated proteins. Since the NMDA receptor performs a critical function at the synapse, it is important to understand how this receptor is delivered to the synapse and how the number and composition of receptors at the synapse are regulated. In addition to being at the synapse, some NMDA receptors are extrasynaptic where they may have functions distinct from those at the synapse. As we reported previously, these two populations are regulated differently. It has been shown previously that NMDA receptors can bind to several PDZ proteins through an interaction with the distal C-terminus of the NR2 subunit. Of particular importance is the PSD-95 family of PDZ proteins, which includes, in addition to PSD-95, PSD-93, SAP97 and SAP102. In our earlier studies, we found that SAP102, an interacting partner of the NMDA receptor, was present throughout the neuron while its companion PDZ protein, PSD-95, was much more restricted to the synapse. Since both associate with the NMDA receptor, this led to the suggestion that SAP102 may be associated with NMDA receptors that are being transported to and from the synapse. Addressing this hypothesis, we carried out a yeast two hybrid screen using the PDZ domain of SAP102 as bait. We previously reported on one of the interacting proteins, Sec8, a component of the exocyst. This past year we characterized another interacting protein, LGN, the mammalian homologue of Drosophila Partner of Inscuteable (Pins). LGN interacts with SH3/GK region of SAP102 through its linker domain. A complex containing LGN, SAP102 and the NMDA receptor can be isolated from mammalian brain. The properties of this interaction were investigated in heterologous cells and in cultured neurons. Expression of LGN in hippocampal neurons mediates trafficking of SAP102 and NMDA receptors to the cell surface. LGN expression also affects the number and size of dendritic spines. This past year we completed a study in which we used chimeras of GFP-tagged temperature-sensitive vesicular stomatitis viral glycoprotein mutant (VSVG) and the distal segment of NR2A or NR2B subunits to study trafficking of NMDA receptors in neurons. As reported last year, while chimeras of NR2A and NR2B are trafficked similarly, their interactions with SAP102 and PSD-95 differ. We find the SAP102 associates with the chimera immediately after release from the ER, which fits very well with our data on the exocyst and SAP102 association, and remains associated until the chimera reaches the synapse. PSD-95, however, has a somewhat different pattern in that it associates at the level of the trans-Golgi network. This past year we completed a study on identifying motifs in the NR2B subunit that are important for its retention in the endoplasmic reticulum (ER). While a specific motif could not be isolated, a segment of the C-terminus was found to be important to its retention. We also found that the motif, HLFY, in the proximal region of the C-terminus, is critical for the exit of the functional, assembled receptor complex from the ER. We investigated motifs within the NR2B C-terminus that are important for the synaptic localization of the NMDA receptor. The PDZ binding domain, -SDV, at the distal end of the C-terminus of the NR2B subunit is necessary for the synaptic localization of NR2B-containing NMDARs. We find that a second domain, the clathrin adaptor protein (AP-2) binding motif, YEKL, also regulates the synaptic localization of receptors. Mutations in this motif can significantly increase the number of synaptic receptors. More importantly, mutations in the AP-2 binding motif allow the synaptic localization of NR2B subunits in the absence of a functional PDZ-binding domain. Fyn kinase can phosphorylate the NR2B subunit within the YEKL motif and potentially inhibit AP-2 binding. We find that expression of a constitutively active Fyn kinase increases the synaptic NMDA response and promotes the synaptic localization of NR2B subunits lacking PDZ-binding domains. Therefore, NMDARs are regulated by a balance between PDZ-protein mediated stabilization and AP-2 mediated internalization. In last year?s report, we presented the results of the loss of the AMPA receptor subunit, GluR2, on the remaining subunits in the hippocampal pyramidal neurons. The absence of this key subunit did not alter the expression of the remaining AMPA receptor subunits, but it did lead to the aberrant formation of receptor complexes. Since the composition of AMPA receptors varies widely throughout the nervous system, we studied a series of different neuronal populations to determine the effect of GluR2 removal on the remaining AMPA receptor subunits. A range of responses was seen. A general observation was that synapses that normally had high levels of GluR2, and thus calcium-impermeable AMPA receptors, showed a decrease in AMPA receptors in the absence of GluR2. Synapses with normally low levels of GluR2 showed no decrease in AMPA receptors, and often showed an increase. Therefore, the response of a synapse to the loss of GluR2 may be related to the calcium flux through AMPA receptors normally seen at that synapse. Clathrin-mediated internalization has emerged as a major mechanism for the regulation of some classes of glutamate receptors from the synapse. This past year we completed a study in which we determined the distribution of clathrin-coated pits and various proteins associated with the clathrin complex. Our results suggest that most clathrin-mediated internalization occurs on the side of the spine complex. The occurrence of clathrin-coated pits decreases significantly with age suggesting that other mechanisms may play a more important role in the regulation of glutamate receptors in adult animals. We characterized the mechanism of removal of integral membrane proteins from the plasma membrane of stereocilia of hair cells. A number of proteins, essential to the function of the hair cell, are located on the stereocilia, but little is known about their delivery and removal. Endo- and exocytotic vesicles are not found within the cytoplasm of the stereocilia indicating that addition and removal must occur at the base of the stereocilium. We chose PMCA2 as a model protein due to its relatively high abundance in stereocilia. Using a series of antibodies and fixation conditions, we found the PMCA2 is found in clusters on stereocilia. We developed an antibody to its extracellular domain and used antibody-feeding to label and track PMCA2 molecules on the stereocilia. Tagged proteins are removed with a half-life estimated to be about 8 hours. In monitoring the removal of PMCA2, we found that labeled proteins were lost first from the apex of the stereocilia. This suggests an organized removal of PMCA2 from the stereocilia beginning with the base and ending with the apex, rather than a random removal. Antibody-tagged proteins are found in organelles within the hair and confined to the apical region of the cell.